Neurodegenerative diseases are among the most pervasive health issues afflicting aging populations of the developed world. A specific subset of these disorders which includes Alzheimer?s disease (AD) and chronic traumatic encephalitis (CTE) are known as tauopathies. In tauopathies, the protein tau that is important for the maintenance of healthy neuronal function forms insoluble neurofibrillary tangles that are characteristic of the pathology. Recently, a novel epitope specific to pathological forms of tau has been identified near the N-terminus of the tau protein. Antibodies specific for this epitope are capable of differentially diagnosing unique tauopathies in cross-sections of human tissue. Unfortunately, antibodies are generally not suitable for targeting molecules in the central nervous system because of their large size and difficulties traversing the blood-brain barrier. The goal of this project is to develop small antibody-like peptides and proteins that can be used for non-invasive imaging of pathological tau protein in living subjects. The hypothesis is that smaller affinity molecules will be better suited for imaging applications and will be more amenable to engineering approaches designed to allow passage through the blood-brain barrier. Three peptide and small protein-based scaffolds have been selected that have previously been successfully engineered for affinity applications. These scaffolds will be used to discover novel agents with high affinity and specificity for the N-terminal epitope of pathological tau. Through collaboration with the group that discovered this novel tau epitope (co-sponsor Kanaan), the complementarity determining regions of their unique tau antibodies will be grafted into the binding loops of small antibody-like scaffolds. In Aim 1, combinatorial libraries will be designed by diversifying the binding loops of each scaffold based on natural antibody diversity and screened using a yeast surface display platform. Next, in Aim 2, lead peptides that strongly and selectively bind to the N-terminal epitope of pathological tau will be advanced to cell culture models of tauopathies and the blood-brain barrier for further engineering and optimization, with the goal of developing imaging agents that recognize aggregated tau protein in vitro and ex vivo. A major effort in this aim will be construction of fusion proteins with polypeptide domains known to facilitate passage through the blood-brain barrier via transcytosis. Finally, in Aim 3, the best affinity molecules identified in Aims 1 and 2 will be assessed for their ability to cross the blood-brain barrier in an in vivo (mouse) model system. Here, pharmacokinetic and tissue distribution studies will be performed. Success in this project will yield novel imaging agents that will have the potential to significantly improve the diagnosis and study of tauopathies in human subjects. Through this fellowship, the applicant will acquire the skills necessary to pursue a career in biologic drug discovery and development. The research training will take place in a multidisciplinary atmosphere at the North Campus Research Complex at the University of Michigan. This facility unites labs from the Colleges of Medicine, Dentistry, Engineering, and Pharmacy and will permit the applicant exposure to a wide range of techniques and ideas.